Blood carries oxygen, nutrients, and physiological signals to every cell in the body, while simultaneously providing immunity and protection against outside pathogens. Yet the same ability of blood to spread sustenance also allows for dissemination of disease, be it cancer cells metastasizing to the liver, Ebola virus ravaging the capillaries, Streptococcus pyogenes liquefying flesh, or HIV eluding detection within the very CD4 cells that aim to eliminate infections.
The universal propensity of pathogens and cancers alike to spread via the blood also creates an opportunity for identification and early detection—allowing physicians to better treat and manage patient care. The evolution of AIDS treatments went hand-in-hand with improvements in nucleic acid diagnostics, from initial reverse-transcription PCR assays to protect the nations' blood supply, to sequencing drug-resistant variants, to RT-PCR quantification of viral load to determine treatment efficacy over time. To date, those infected have not been cured, but sophisticated diagnostic tools have guided treatment, epidemiological, and political decisions to stem this global epidemic.
Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries. Cancer has now become the biggest cause of mortality worldwide, with an estimated 8.2 million deaths from cancer in 2012. Cancer cases worldwide are forecast to rise by 75% and reach close to 25 million over the next two decades. A recent report by the world health organization concludes: “(The) Global battle against cancer won't be won with treatment alone. Effective prevention measures (are) urgently needed to prevent (a) cancer crisis”. Detection of early cancer in the blood is the best means of effective prevention. It will save lives by enabling earlier and better treatment, as well as reduce the cost of cancer care.
Plasma or serum from a cancer patient contains nucleic acids released from cancers cells undergoing abnormal physiological processes. These nucleic acids have already demonstrated diagnostic utility (Diaz and Bardelli, J Clin Oncol 32: 579-586 (2014); Bettegowda et al., Sci Transl Med 6: 224 (2014); Newman et al., Nat Med 20: 548-554 (2014); Thierry et al., Nat Med 20: 430-435 (2014)). A further source of nucleic acids is within circulating tumor cells (CTCs), although early stage and a significant fraction of localized tumors send out very few to no CTC's per ml. Normal plasma or serum contains nucleic acids released from normal cells undergoing normal physiological processes (i.e. exosome secretion, apoptosis). There may be additional release of nucleic acids under conditions of stress, inflammation, infection, or injury.
The challenge to develop reliable diagnostic and screening tests is to distinguish those markers emanating from the tumor that are indicative of disease (e.g., early cancer) vs. presence of the same markers emanating from normal tissue (which would lead to a false-positive signal). There is also a need to balance the number of markers examined and the cost of the test, with the specificity and sensitivity of the assay. Comprehensive molecular profiling (mRNA, methylation, copy number, miRNA, mutations) of thousands of tumors by The Cancer Genome Atlas Consortium (TCGA), has revealed that colorectal tumors are as different from each other as they are from breast, prostrate, or other epithelial cancers (TCGA “Comprehensive Molecular Characterization of Human Colon and Rectal Cancer Nature 487: 330-337 (2014)). Further, those few markers they share in common (e.g., KRAS mutations,) are also present in multiple cancer types, hindering the ability to pinpoint the tissue of origin. For early cancer detection, the nucleic acid assay should serve primarily as a screening tool, requiring the availability of secondary diagnostic follow-up (e.g., colonoscopy for colorectal cancer).
Compounding the biological problem is the need to reliably quantify mutation, promoter methylation, or DNA or RNA copy number from either a very small number of initial cells (i.e. from CTCs), or when the cancer signal is from cell-free DNA (cfDNA) in the blood and diluted by an excess of nucleic acid arising from normal cells, or inadvertently released from normal blood cells during sample processing (Mateo et al., Genome Biol 15: 448 (2014)).
Likewise, an analogous problem of identifying rare target is encountered when using nucleic-acid-based techniques to detect infectious diseases directly in the blood. Briefly, either the pathogen may be present at 1 or less colony forming units (cfu)/ml, and/or there are many potential pathogens and sequence variations responsible for virulence or drug resistance. While these issues are exemplified with cancer, it is recognized that the solutions are equally applicable to infectious diseases.
A Continuum of Diagnostic needs Require a Continuum of Diagnostic Tests.
The majority of current molecular diagnostics efforts in cancer have centered on: (i) prognostic and predictive genomics, e.g., identifying inherited mutations in cancer predisposition genes, such as BrCA1, BrCA2, (Ford et al. Am J Hum Genet 62: 676-689 (1998)) (ii) individualized treatment, e.g., mutations in the EGFR gene guiding personalized medicine (Sequist and Lynch, Ann Rev Med, 59: 429-442 (2008), and (iii) recurrence monitoring, e.g., detecting emerging KRAS mutations in patients developing resistance to drug treatments (Hiley et al., Genome Biol 15: 453 (2014); Amado et al., J Clin Oncol 26: 1626-1634 (2008)). Yet, this misses major opportunities in the cancer molecular diagnostics continuum: (i) more frequent screening of those with a family history, (ii) screening for detection of early disease, and (iii) monitoring treatment efficacy. To address these three unmet needs, a new metric for blood-based detection termed “cancer marker load”, analogous to viral load is herein proposed.
DNA sequencing provides the ultimate ability to distinguish all nucleic acid changes associated with disease. However, the process still requires multiple up-front sample and template preparation, and is not always cost-effective. DNA microarrays can provide substantial information about multiple sequence variants, such as SNPs or different RNA expression levels, and are less costly then sequencing; however, they are less suited for obtaining highly quantitative results, nor for detecting low abundance mutations. On the other end of the spectrum is the TaqMan™ reaction, which provides real-time quantification of a known gene, but is less suitable for distinguishing multiple sequence variants or low abundance mutations.
It is critical to match each unmet diagnostic need with the appropriate diagnostic test—one that combines the divergent goals of achieving both high sensitivity (i.e., low false-negatives) and high specificity (i.e., low false-positives) at a low cost. For example, direct sequencing of EGFR exons from a tumor biopsy to determine treatment for non-small cell lung cancer (NSCLC) is significantly more accurate and cost effective than designing TaqMan™ probes for the over 180 known mutations whose drug response is already catalogued (Jia et al. Genome Res 23: 1434-1445 (2013)). The most sensitive technique for detecting point mutations, BEAMing (Dressman et al., Proc Natl Acad Sci USA 100: 8817-8822 (2003)), rely on prior knowledge of which mutations to look for, and thus are best suited for monitoring for disease recurrence, rather than for early detection. Likewise, to monitor blood levels of Bcr-Abl translocations when treating CML patients with Gleevec (Jabbour et al., Cancer 112: 2112-2118 (2008)), a simple quantitative reverse-transcription PCR assay is far preferable to sequencing the entire genomic DNA in 1 ml of blood (9 million cells×3 GB=27 million Gb of raw data).
Sequencing 2.1 Gb each of cell-free DNA (cfDNA) isolated from NSCLC patients was used to provide 10,000-fold coverage on 125 kb of targeted DNA (Kandoth et al. Nature 502: 333-339 (2013)). This approach correctly identified mutations present in matched tumors, although only 50% of stage 1 tumors were covered. The approach has promise for NSCLC, where samples average 5 to 20 mutations/Mb, however would not be cost effective for other cancers such as breast and ovarian, that average less than 1 to 2 mutations per Mb. Current up-front ligation, amplification, and/or capture steps required for highly accurate targeted deep sequencing are still more complex than multiplexed PCR-TaqMan™ or PCR-LDR assays.
A comprehensive data analysis of over 600 colorectal cancer samples that takes into account tumor heterogeneity, tumor clusters, and biological/technical false-positives ranging from 3% to 10% per individual marker showed that the optimal early detection screen for colorectal cancer would require at least 5 to 6 positive markers out of 24 markers tested (Bacolod et al., Cancer Res 69:723-727 (2009); Tsafrir et al. Cancer Res 66: 2129-2137 (2006), Weinstein et al., Nat Genet 45: 1113-1120 (2013); Navin N.E. Genome Biol 15: 452 (2014); Hiley et al., Genome Biol 15: 453 (2014)); Esserman et al. Lancet Oncol 15: e234-242 (2014)). Further, marker distribution is biased into different tumor clades, e.g., some tumors are heavily methylated, while others are barely methylated, and indistinguishable from age-related methylation of adjacent tissue. Consequently, a multidimensional approach using combinations of 3-5 sets of mutation, methylation, miRNA, mRNA, copy-variation, alternative splicing, or translocation markers is needed to obtain sufficient coverage of all different tumor clades. Analogous to non-invasive prenatal screening for trisomy, based on sequencing or performing ligation detection on random fragments of cfDNA (Benn et al., Ultrasound Obstet Gynecol. 42(1):15-33 (2013); Chiu et al., Proc Natl Acad Sci USA 105: 20458-20463 (2008); Juneau et al., Fetal Diagn Ther. 36(4) (2014)), the actual markers scored in a cancer screen are secondary to accurate quantification of those positive markers in the plasma.
Technical Challenges of Cancer Diagnostic Test Development.
Diagnostic tests that aim to find very rare or low-abundance mutant sequences face potential false-positive signal arising from: (i) polymerase error in replicating wild-type target, (ii) DNA sequencing error, (iii) mis-ligation on wild-type target, (iii) target independent PCR product, and (iv) carryover contamination of PCR products arising from a previous positive sample. The profound clinical implications of a positive test result when screening for cancer demand that such a test use all means possible to virtually eliminate false-positives.
Central to the concept of nucleic acid detection is the selective amplification or purification of the desired cancer-specific markers away from the same or closely similar markers from normal cells. These approaches include: (i) multiple primer binding regions for orthogonal amplification and detection, (ii) affinity selection of CTC's or exosomes, and (iii) spatial dilution of the sample.
The success of PCR-LDR, which uses 4 primer-binding regions to assure sensitivity and specificity, has previously been demonstrated. Desired regions are amplified using pairs or even tandem pairs of PCR primers, followed by orthogonal nested LDR primer pairs for detection. One advantage of using PCR-LDR is the ability to perform proportional PCR amplification of multiple fragments to enrich for low copy targets, and then use quantitative LDR to directly identify cancer-specific mutations. Biofire/bioMerieux has developed a similar technology termed “film array”; wherein initial multiplexed PCR reaction products are redistributed into individual wells, and then nested real-time PCR performed with SYBR Green Dye detection.
Affinity purification of CTC's using antibody or aptamer capture has been demonstrated (Adams et al., J Am Chem Soc 130: 8633-8641 (2008); Dharmasiri et al, Electrophoresis 30: 3289-3300 (2009); Soper et al. Biosens Bioelectron 21: 1932-1942 (2006)). Peptide affinity capture of exosomes has been reported in the literature. Enrichment of these tumor-specific fractions from the blood enables copy number quantification, as well as simplifying screening and verification assays.
The last approach, spatial dilution of the sample, is employed in digital PCR as well as its close cousin known as BEAMing (Vogelstein and Kinzler, Proc Natl Acad Sci US A. 96(16):9236-41 (1999); Dressman et al., Proc Natl Acad Sci USA 100: 8817-8822 (2003)). The rational for digital PCR is to overcome the limit of enzymatic discrimination when the sample comprises very few target molecules containing a known mutation in a 1,000 to 10,000-fold excess of wild-type DNA. By diluting input DNA into 20,000 or more droplets or beads to distribute less than one molecule of target per droplet, the DNA may be amplified via PCR, and then detected via probe hybridization or TaqMan™ reaction, giving in essence a 0/1 digital score. The approach is currently the most sensitive for finding point mutations in plasma, but it does require prior knowledge of the mutations being scored, as well as a separate digital dilution for each mutation, which would deplete the entire sample to score just a few mutations.
Real-time PCR & Microfluidic Instrumentation
A number of PCR assays/microfabricated devices have been designed for rapid detection of pathogens and disease-associated translocations and mutations. Each assay/hardware combination has particular strengths, but when combined with the real world problem of multidimensional and multiplexed markers required for cancer detection, the flexibility of PCR-LDR with microfluidics provides certain advantages.
Instrumentation, assay design, and microfluidic architecture need to be seamlessly integrated. Some PCR instrumentation use real-time fluorescence or end-point fluorescence to quantify initial template molecules by cycling chambers, wells, or droplets through different temperatures. Yet other instrumentation comprises addressable microfluidic plates for real-time PCR detection. However the high cost of both the instruments and consumables has limited the widespread use of these machines for clinical applications.
In a different architecture, termed continuous-flow PCR, the reaction mix moves through channels that are neatly arranged in a radiator pattern, and flow over heating elements that are at fixed temperatures. This architecture allows the entire amplification reaction to be completed in a few minutes, and is ideal for capillary separation and readout. For ligase detection reactions, the readout may be achieved by taking advantage of LDR-FRET or electronic detection. In LDR-FRET, one primer has a donor, the other has an acceptor group, and after ligation they form a hairpin. This allows for counting single ligation events to obtain highly quantitative readouts of input DNA copy number. Alternatively, by appending gold-nanoparticles on each primer, the ligation product will contain two nano-particles, and these may be distinguished using electronic readout.
In considering various degrees of automation, the approach described herein is guided by the principles of “modularity” and “scalability”. Firstly, the process should be separated into modular steps that may initially be optimized on separate instruments. For example, the device may be comprised of a first module for purification of DNA from plasma cfDNA as well as RNA from exosomes, a second module for multiplexed reverse transcription and/or limited amplification of various targets, and a third module for generating and detecting ligation products. Such a modular architecture allows for swapping in improved modules that keep pace with technological developments. For the modularity approach to work, it is critical that products from one module can be moved seamlessly into the next module, without leakage and without worry of crossover contamination.
Secondly, the modular design should be amenable to scalable manufacture in high volumes at low cost. The manufacturing costs and how primers/reagents/samples are deposited into the device must be taken into consideration.
The present invention is directed at overcoming these and other deficiencies in the art.